**2.1 Improving stress tolerance**

*S. cerevisiae* must deal with different stress conditions, osmotic stress due to the high levels of sugars, oxidative stress, low nitrogen levels and high levels of ethanol among others. These stresses can produce problems in the wine fermentation process [7]. One way to solve these problems is to use engineering yeast strains that can grow better in these conditions.

The main component in the grape juice is monosaccharides (glucose + fructose) and their total concentration vary between 170 and 220 g/L [2] but can be up to 340 g/L. This extremes levels of sugars can inhibit yeast growth because of the osmotic pressure, that is called hyperosmotic stress. High Osmolarity Glycerol response (HOG) is the pathway that are regulated the response against osmotic stress, inducing the gene expression for glycerol production (*GPD1* and *GPD2*) and for glycerol uptake (*STL1*) [8]. Deletion of Stl1 (glycerol symporter) has a slower growth in ice wine juice and elevate glycerol and acetic acid production, so these genes could be a target to improve these conditions [9].

Aerobic organisms depend on oxygen in cellular respiration but at high concentrations its oxidant power produces cytotoxic compounds called reactive oxygen species (ROS) that are unstable oxygen species with unpaired electrons that if they are not remove from the cell can damage macromolecules as DNA, proteins and lipids. It is during the active dry yeast production (ADY) where the yeast is in a higher oxidative stress condition. For example, oxidative stress-related genes (as thioredoxines, glutaredoxins and peroxiredoxins) are induced during this process [10]. Overexpression of the cytosolic thioredoxin 2 gene, *TRX2*, leads a wine yeast

#### *Genetically Modified Yeasts in Wine Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.98639*

increase biomass production [11]. This ADY process cause an internal oxidative stress and there are molecules and enzymes that helps to reduce the oxidative stress as glutathione (GSH), trehalose, catalase, superoxide dismutase and glutathione reductase [12]. For example, deletion of the main cytosolic peroxiredoxin, Tsa1, in the industrial wine yeast L2056 increase trehalose and glycogen accumulation playing a role in the regulation of metabolic reactions that are important for the final product [13]. Moreover, overexpression of superoxide dismutase 1 and 2 (*SOD1* and *SOD2*) and *HSP12* (a plasma membrane protein involved in maintaining membrane organization) genes improves vellum formation and cell viability in three strains of Sherry flor yeast, and improve in the specific activities and higher levels of GSH peroxidase and glutathione reductase activities and higher intracellular concentrations of GSH and lower peroxidized lipid concentration [14]. Moreover, an indigenous strain of *S. cerevisiae* called RIA with the insertion thought homologs recombination of ilv2Δ::GSH1-*CUP1* improves glutathione production (19%) with the same fermentation capacity than the wild type [15].

At the end of the fermentation process, there are high levels of ethanol (11–14%). The toxicity of ethanol inhibited glucose and amino acid uptake because ethanol damage cell membranes [2, 16]. Overexpression of *TPS1* (synthase subunit of trehalase-6-P synthase/phosphatase complex) and deletion of *NTH1* (neutral trehalose) increase ethanol tolerance [17]. Besides, overexpression of *GSY2* (Glycogen synthase) and *NTH1* increased respectively glycogen and trehalose levels that are important for the fermentative capacity [18]. Global transcription machinery engineering (gTME) is a technique that alter key proteins to regulate the global transcriptome by error-prone polymerase chain reaction (epPCR) mutations. With this technique, a *SPT15* (TATA binding protein) mutagenesis strains was constructed with a higher ethanol tolerance [19] and it was found that the mutant of the *SPT8* (SAGA complex) gave 8.9% higher ethanol tolerance [20]. Direct evolution method was performed to engineer RNA polymerase II (RNAPII) subunit 7 (which plays a central role in mRNAs synthesis) in the yeast strain (M1) that improved ethanol titer and improved other stress as osmotolerance [21].

Some species of *Saccharomyces* genus have shown better adaptation at low temperatures than *cerevisiae*, which was the case of cryotolerant yeast *S. uvarum* and *S.* k*udriavzevii*. This better cold adaptation is because the higher amount of proteins related with translation (more ribosomes proteins in psychrotolerant strains) and the importance of the oxidative stress response in the adaptation of cold fermentation (mutants in *AHP1*, *MUP1* and *URM1* has a strongly impaired low-temperature growth) [22, 23]. Recently, Ying Su *et al* noticed that the hybrids low nitrogendemanding cryotolerant *S. eubayanus* and *S uvarum* conferred better fermentations rates under low temperature or low-nitrogen conditions [24].
